1. Field of the Invention
This invention relates to a process for the purification of enzyme preparations that are highly adsorbed by ion exchange materials. More specifically the invention relates to a process for the purification of enzyme preparations by a chromatographic separation.
2. Description of the Prior Art
Many industrial enzymes of biological origin are utilized in commercial food processes. Enzymes of the amylase type are produced in larger quantities and their value exceeds all the other enzyme preparations produced in the U.S. For example, alpha amylase, beta amylase, and glucoamylase enzymes are widely used in processes to convert starch to glucose, and in the brewing, distilling and baking industries. Glucose isomerase is used to convert glucose to fructose. Invertase is used in the manufacture of liquid and soft centered candies, and for the conversion of sucrose to invert sugar. The invert sugar is used in production of confectionary, cordials, ice cream, and soft drinks. Lactase is used in the manufacture of ice cream and in whey to produce fermentable sugars.
Pectic and protease enzymes are also utilized commercially. Pectic enzymes are used in production of fruit juices and fruit juice products, wines, fermentation of coffee and cocoa beans, and in the rehydration of dehydrated foods. Proteases are used in cheese making, meat tenderizing, bread baking, for haze elimination from beer and other beverages, and in the preparation of digestive aids.
Enzyme preparations are produced by the cultivation of selected strains of microorganisms and are predominantly used in the soluble form. However, in addition to the desirable enzyme, the microorganisms also produce a wide variety of biochemical compounds required for growth and productivity. As a result, soluble enzyme preparations, whether obtained from the extracellular medium or extracted from the microbial cells, usually contain many undesirable impurities. The insoluble impurities can be easily separated by well known methods, such as by filtration or centrifugation. However, the soluble impurities are difficult and expensive to remove because they often have chemical or physical properties similar to the desired product. The enzyme cost is the major factor in determining its commercial acceptability. To minimize enzyme cost, industrial enzyme preparations are purified only to the extent necessary for the desired efficacy in the intended use. As a result, enzymes for industrial use are usually not treated to remove soluble impurities.
One of the major technological advancements in recent years has been the development of processes to produce immobilized (insoluble) enzymes. The immobilized enzymes are particularly adaptable to continuous processes which are more economical than batch processes. For example, immobilized glucose isomerase is utilized in continuous processes to isomerize glucose to fructose in the commercial production of high fructose corn syrup. The production of other immobilized enzymes, such as glucoamylase, invertase, and lactase have also been reported but have not found widespread commercial use.
Glucose isomerase is an intracellular enzyme produced by submerged aerobic fermentation of a selected microorganism. For example, microorganisms of the genera Actinoplanes, Arthrobacter, Lactobacillus and Streptomyces produce intracellular glucose isomerase. Glucose isomerase may be immobilized in the presence of the microbial cells, for example, by a chemical treatment, or the enzyme may be extracted from the cells and separated as a soluble enzyme prior to immobilization on an inert carrier. Both procedures are utilized to produce immobilized enzyme used in the production of high fructose corn syrup.
A procedure for immobilization of glucose isomerase within the microbial cells by a heat treatment is disclosed in U.S. Pat. No. 3,753,858--Takasaki et al. U.S. Pat. No. 3,779,869--Zienty and U.S. Pat. No. 3,980,521--Amotz et al., disclose the immobilization of glucose isomerase with the microbial cells by treatment with gluteraldehyde. U.S. Pat. No. 3,821,086--Lee et al. and U.S. Pat. No. 3,935,069--Long, disclose immobilized glucose isomerase produced by a flocculation of the microbial cells.
Processes for immobilization of a cell-free, soluble glucose isomerase of inert carriers are disclosed in a number of U.S. patents. U.S. Pat. No. 3,708,397--Sipos, U.S. Pat. Nos. 3,788,943 and 3,909,354 both to Thompson et al., and U.S. Pat. Nos. 3,960,663--Tamura et al., disclose methods for immobilizing a soluble cell-free glucose isomerase on an anion exchange cellulose or a synthetic anion exchange resin. U.S. Pat. Nos. 3,850,751 and 3,868,304 both to Messing disclose processes for the immobilization of soluble, cell-free glucose isomerase on a porous ceramic body and a porous alumina body, respectively. U.S. Pat. No. 3,715,277--Dinelli discloses a method for the immobilization of soluble, cell-free glucose isomerase by entrapment in a polymeric fiber.
Glucose isomerase is produced primarily intracellularly and thus the major portion of the glucose isomerase is found within and/or on the cell walls of the microorganisms. Therefore, it is necessary to extract the enzyme from the cells to produce the soluble enzyme. The extraction process, which makes use of a cationic surfactant or other agent, results in at least partial disruption of the cell envelope allowing diffusion of the enzyme and other cellular materials into the extraction medium. After extraction and removal of the insoluble debris, te enzyme is immobilized directly on an insoluble carrier. Typically, the enzyme is adsorbed on an anion exchange matrix such as DEAE-cellulose or a granular ion exchange resin.
The amount of enzyme activity adsorbed on an ion exchange material is not dependent on the concentration of enzyme in enzyme extract, so long as the amount of total activity supplied is sufficient to satisfy the total capacity of the adsorbent. However, the amount of enzyme activity adsorbed is a function of the purity or quality of the soluble enzyme extract. That is, increasing the enzyme purity will result in an increase in the enzyme activity per gram of adsorbent. This is because the soluble impurities may interfere with the enzyme adsorption, or may compete with the enzyme for adsorption on the available ion exchange sites of the insoluble matrix. As a result, ion exchange sites occupied by impurities will not be available for binding of active enzyme. The impurities which compete for the ion exchange sites are believed to be charged biological oligomers or polymers, e.g., nucleic acids, proteins, etc. Removal of those substances which compete with the enzyme for adsorption sites can result in improved adsorption of the enzyme and the resulting immobilized enzyme will have a substantially higher activity per gram. Higher activity is of particular importance when the cost of the matrix is high. Therefore, an economical process to remove the soluble impurities would be desirable.
Methods for removal or separation of undesirable materials for biological extracts are well known. A current summary of these methods can be found in Volume XXII of "Methods in Enzymology" pp. 273-287 and pp. 476-556 (ed. W. E. Jakoby, Academic Press, N.Y., N.Y.). Various separation methods for enzyme purification, such as separation based on solubility, separation based on specific affinity and chromatographic separations are described.
Column chromatography is a widely used laboratory technique for enzyme purification. In this method, the enzyme is adsorbed on an ion exchange material, such as DEAE-cellulose or CM-cellulose, while the impurities remain in the effluent from the column. The enzyme is then eluted from the adsorbent by the addition of a solution containing an agent, such as a salt, to affect a change in ionic strength or pH. On a laboratory scale, this type of separation produces a highly purified product with good recovery of the total enzyme activity. However, the agent used to elute the enzyme from the ion exchange adsorbent interferes with the subsequent enzyme immobilization. Therefore, the eluting agent must be removed before the purified enzyme can be efficiently immobilized on an insoluble carrier. The added cost of this separation step is relatively unimportant in a laboratory process where the goal is to produce a highly purified enzyme. However, cost is important in commercial use where the goal is to produce an enzyme at a minimum cost which will produce economically acceptable results. The need for careful control of operating conditions is also a drawback. Thus, commercial use of a chromatographic separation or refining process is limited by the overall cost, which may outweigh the benefits.
Numerous patents also describe various methods for purification of enzymes. U.S. Pat. No. 3,769,168 to Masuda describes the purification of beta amylase by adsorption, washing and eluting the enzyme with an ionic solution. U.S. Pat. No. 3,912,595 to Philipp et al. describes the purification of a hydrolytic enzyme solution by reversibly complexing the enzyme on a granular support material in a column, after which the enzyme is recovered by elution with a buffer. U.S. Pat. No. 3,972,777 to Yamada et al. describes a method to refine .alpha.-galactosidase by selective adsorption on an acidic cation exchange resin and then eluting the .alpha.-galactosidase from the resin with a buffer. All of these methods encompass contacting an impure enzyme solution with a matrix which will adsorb or bond the enzyme, and then eluting the purified enzyme from the matrix by addition of an ionic solution. The cost of elution and recovery of the purified enzyme has been a deterrent to achieving commercial acceptability.
In U.S. Pat. No. 4,106,992 to Vairel et al., crude urokinase is subjected to exclusion chromatography utilizing a DEAE-cellulose resin. The described process is principally directed to removing pyrogenic substances from urokinase. The disadvantages are that it is a cumbersome process and requires conductivity and pH conditions which are critical to achieve the desired results. Salts, such as ammonium sulfate, are utilized to achieve the required conductivity.
U.S. Pat. No. 4,055,469 to Snoke et al. describes the removal of nucleic acids and unwanted proteins from microbial extracts by precipitation.
U.S. Pat. Nos. 3,788,945 and 3,909,354 both to Thompson et al. describes a batch method for purification of a glucose isomerase solution by contacting it with DEAE-cellulose for about 30 minutes, after which the DEAE-cellulose was removed by filtration, the filter cake washed with water and the washings were collected with the enzyme containing filtrate. This process results in dilution of the enzyme preparation. The amount of DEAE-cellulose used in the batch process of Thompson is critical. If an insufficient amount of DEAE-cellulose is used, the maximum purification of the enzyme will not be achieved. If too much DEAE-cellulose is used, some enzyme will be adsorbed and retained on the absorbent resulting in a loss of activity in the purified enzyme.
A description of chromatographic methods contained in the "Encyclopedia of Chemical Technology", Volume 5, pp. 418-420 (Kirk Othmer, 2nd Ed.; Wiley-Interscience, N.Y., N.Y.) includes a method referred to as "Frontal Analysis". The "Frontal Analysis" technique is generally similar to the process of our invention. However, the novelty of our invention is the discovery that ionic biological impurities in an enzyme solution can be used to displace enzyme from an adsorbent using a frontal analysis process.
Throughout this specification and claims, "activity" is defined as units of activity per ml when reference is made to an enzyme solution. "Activity" is defined as units per gram, d. b. when reference is made to an immobilized or dry enzyme preparation.